Preliminary Studies Of Identification, Inhibitory Characteristics And Application Of An Antagonistic Bacterium Strain Against Vibrio

In recent years, with the rapid development of aquaculture, the animals are threatened by diseases more serously. The occurrence of diseases brings not only huge economic losses but also great impact on the environment at the same time. The abuse of chemical drugs such as antibiotics which are used for preventing the disease has caused serious harm on the environment and human. As substitutes for antibiotics, probiotics can inhibit pathogenic bacteria, provide nutritions for animals, enhance the ability of immunity of animals, improve the water quality conditions and repair the ecological environment. So the usage of probiotics in aquaculture is attracting more attention gradually. One of the inhibitory mechanisms of probiotics is to restrain the growth of the pathogen bacteria by producing inhibitory substances. Therefore, it is important to choose some bacterias which have great inhibitory activity as potential probiotics.In this study, we did some researches on the indentification, inhibitory substances and application of an antagonistic bacterium strain against vibrio. It is aimed to provide a theoretical basis for its applicaton in aquaculture. The results of the study are as follows:1. Identification of an antagonistic bacterium strain against vibrio and study of its inhibitory activityIn this experiment, we separated and screened an antagonistic bacterium strain, named it as BL1. After tested by cross-streaking method, spot inoculation and well test, it is found that strain BL1 has obvious inhibitory activity against Vibrio alginolyticus, V. vulnificus and V. parahaemolyticus. According to the results of the morphological, physiological and 16S rDNA sequence analysis, strain BL1 is identified as Bacillus subtilis. The single-factor analysis further helps to confirm the best culture conditions of strain BL1 is 28℃, pH7.0, inoculation quantity of 1% (V/V) and 190rpm/min.2. Separation and characterization of inhibitory substances of strain BL1Taking the fermentation liquor of BL1 as the subject, Vibrio vulnificus as the indicator bacteria, the characterization of inhibitory substances are premilinary studied through well test. First, the experiment about effect of time on the inhibitory activity shows that 24h is the best. Second, after preliminary purification including filter, ammonium sulfate precipitation and ultra filtration, it is found that the inhibitory substances are extracellular, non-protein and have greater molecular weight than 10 kDa. At last, the inhibitory substances have great ability of enduring the changes of environment, such as temperature (60-100℃) and pH (3.0-11.0). And they are not sensitive to proteinase K. The results show the inhibitory substances produced by strain BL1 have great inhibitory ability and stability, which indicates strain BL1 can be used as potential probiotic in aquaculture.3. Effects of strain BL1 on the growth of Litopenaeus vannamei and the quantity of vibrio in water.The safety inspection proves that strain BL1 has no negative effects on litopenaeus vannamei, therefore, strain BL1 is applied to the water to study its impact on growth and the quantity of vibrio in water. The additive concentrations of living bacillus are 107,109 and 1011 cfu/kg for supplement in feed (T1, T2 and T3)and 106,108 and 1010 cfu/m3 for addition to water (P1, P2 and P3) respectively. The control was feed with basic bait without BL1 (A). The experiment lasts 42 days. The quantity of vibrio in water is determined at the time of 0.5,1,2,3,4 and 5d of the cycle. At the end of the experiment, measure the weight growth rate (WGR), specific growth rate (SGR), feed utilization rate (FER) and survival rate (SR). The results show that:(1) in the respect of growth, strain BL1 in all the treatment groups except P1 can significantly increases SGR, promotes the growth of the shrimps. While the T2 (109 cfu/kg) is the best, P2 and P3 (108 and 1010 cfu/m3) have no significantly different effect on SGR and FER (P>0.05); (2) all the different dose of strain BL1 for supplement in feed can reduce the quantity of vibrio effectively and permanently, and they have the similar change rule. The quantity of vibrio of P1, P2 and P3 are also lower than that of the control group during experiment, and the effect is better with the concentration increasing. Finally, in the water of the P3 (1010 cfu/m3), the quantity of vibrio falls to the lowest level. But, the study also finds that the quantity of vibrio in addition to water group will partly rebound on 3-5d.